C. elegans, we identified a mutation in muscleblind-1/mbl-1 as a suppressor of loss of kinesin-13 family microtubule destabilizing factor klp-7. Muscleblind-1(MBL-1) is an RNA-binding protein that regulates the splicing, localization, and stability of RNA. Our findings demonstrate that mbl-1 is required cell-autonomously for axon growth and proper synapse positioning in the posterior lateral microtubule (PLM) neuron. Loss of mbl-1 leads to increased microtubule dynamics and mixed orientation of microtubules in the anterior neurite of PLM. These defects are also accompanied by abnormal axonal transport of the synaptic protein RAB-3 and reduction of gentle touch sensation in mbl-1 mutant. Our data also revealed that mbl-1 is genetically epistatic to mec-7 (β tubulin) and mec-12 (α tubulin) in regulating axon growth. Furthermore, mbl-1 is epistatic to sad-1, an ortholog of BRSK/Brain specific-serine/threonine kinase and a known regulator of synaptic machinery, for synapse formation at the correct location of the PLM neurite. Notably, the immunoprecipitation of MBL-1 resulted in the co-purification of mec-7, mec-12, and sad-1 mRNAs, suggesting a direct interaction between MBL-1 and these transcripts. Additionally, mbl-1 mutants exhibited reduced levels and stability of mec-7 and mec-12 transcripts. Our study establishes a previously unknown link between RNA-binding proteins and cytoskeletal machinery, highlighting their crucial roles in the development and maintenance of the nervous system.Regulation of the microtubule cytoskeleton is crucial for the development and maintenance of neuronal architecture, and recent studies have highlighted the significance of regulated RNA processing in the establishment and maintenance of neural circuits. In a genetic screen conducted using mechanosensory neurons of  C. elegans, we have uncovered a molecular connection between the RNA binding protein Muscleblind-1 (MBNL) and the microtubule cytoskeleton, specifically in neurons responsible for gentle touch sensation. Our investigations have revealed that Muscleblind-1/MBL-1 is involved in the optimal expression of the mRNAs that encode for the building blocks of microtubules, namely, the tubulin subunits MEC-7 (β-tubulin) and MEC-12 (α-tubulin) in touch neurons. Additionally, MBL-1 binds to another mRNA, sad-1, which encodes a kinase protein essential for synapse formation. In the absence of mbl-1, the microtubule cytoskeleton becomes unstable, leading to significant disruptions in the transport system within the neurite. Consequently, this impairment results in a reduction of sensory function performed by this set of neurons. The findings from our study provide a mechanistic insight into how RNA-binding proteins and the microtubule cytoskeleton are linked in neurons.Both RNA-binding proteins and microtubule cytoskeleton play important roles in the development and maintenance of a functional nervous system. A misregulation of mRNA processing and microtubule dynamics often contributes to neurodevelopmental and neurodegenerative disorders. Through a combination of genetic approaches and live cell imaging in the nematode  A highly ordered functional neuronal circuit comprises polarized nerve cells, which are compartmentalized into dendrites and axons for unidirectional reception and transmission of information. Several reports suggest that the structural and functional polarity of neurons relies on cytoskeletal elements within the neuron –4. TheseIn vertebrate neurons, axonal microtubules are arranged in a parallel array with their plus-ends facing toward the synapse, while dendrites exhibit a mixed orientation of microtubules . In inveRecent reports have identified the critical roles of RNA-binding proteins in neuronal development  and synaDrosophila showed that Muscleblind (MBL) is expressed in the nervous system and regulates alternative splicing of Dscam2 during the development of the nervous system .Immunoprecipitation experiments were conducted following previously described protocols . ApproxiFP::FLAG ,97 or PmThe homogenized sample was passed successively through 19 mm, 22 mm, 26 mm, and an Insulin syringe to make a smooth homogenate, which was then centrifuged, at 19,000Xg for 20 minutes, to obtain a clear supernatant. A portion (5–10%) of the total lysate was set aside as the total input for RNA estimation and western blotting. The total protein concentration in each sample was determined using the Bradford assay. Equal amounts of protein were used across all conditions for the RIP experiments.Agarose beads  were equilibrated in 1X lysis buffer. For pre-clearing, each supernatant was incubated with 20 μl of equilibrated agarose beads for one hour, followed by centrifugation at 8,000Xg for 10 minutes to collect the beads. The supernatant obtained after this step was the pre-cleared lysate, which was further processed.Pmec-4::MBL-1::GFP from wild-type and mbl-1 samples expressing this transgene, 3 μg of anti-GFP antibody  was added to the precleared lysates and incubated for 8–10 hours with continuous mixing. The following day, 30 μl of equilibrated Protein G–agarose beads (Sigma 15920010) were added to the lysates and incubated further for 3 hours.To immunoprecipitate Flag-tagged GFP from the strains, anti-Flag M2 agarose beads  were used. For each sample 60 μl of 50% slurry  of anti-Flag M2 agarose beads was taken in a 1.5 ml tube and washed with 1X lysis buffer to equilibrate. The pre-cleared lysates from the previous step were added to the equilibrated beads and incubated at 4°C overnight with continuous mixing. In the case of immunoprecipitation of Both samples (anti-Flag M2 and anti-GFP) were then centrifuged at 10,000 rpm for 15 minutes at 4°C. The resulting pellets were collected and washed with lysis buffer containing 150 mM NaCl, followed by centrifugation at 10,000Xg for 15 minutes at 4°C. The agarose beads were collected, with 20% of the bead’s volume reserved for western Blot analysis and the remaining 80% was used for RNA isolation and qRT PCR as described earlier.Samples were boiled in Laemmli buffer and resolved on a 12% SDS-PAGE. After the proteins were transferred to nitrocellulose membranes, the membranes were blocked with 5% BSA for one hour and then probed with the anti-GFP antibody (Abcam ab290) overnight. The following day, the membranes were washed in 1X Tris Buffer Saline containing 0.1% Tween 20 (TBST) and then incubated with a secondary antibody (anti-Rb) for 3 hours. Finally, the protein bands were detected using a standard ECL chemiluminescence detection kit .Pmec-4::mCherry (tbIs222) along with either Pmec-7::mec-7::gfp  or Pmec-7::mec-7::gfp were imaged using 10% of 488 nm laser for GFP tagged cDNA, 1.5% of 488nm laser for GFP tagged genomic DNA, and 0.3% of 561 nm laser for mCherry (constitutive reporter).For image acquisition and quantification of fluorescent intensities a Nikon confocal microscope (A1HD25) with a 60X oil objective (NA 1.4) was used. The worms were mounted on 5% agarose beds and immobilized using 10 mM levamisole in M9 buffer. The transgenic worms co-expressing mec-7::gfp, or mec-12::gfp, and mCherry reporters were measured from the PLM cell body and the 50 μm regions of both the anterior and posterior neurite of PLM neuron, using ImageJ. We measured average intensities from ROIs placed on the background, outside the PLM neuron for background correction. The ratio of average intensities of GFP and MCherry ::gfp::SL2::mCherry, which co-expresses the mec-7::gfp (genomic DNA) and mCherry under the same promoter,) were imaged using 8% of 488 nm laser for GFP and 1% of 561 nm laser for mCherry from the same ROIs as discussed above.Similarly, worms expressing gfp::mec-12 and mCherry  and Pmec-4::mCherry (tbIs222) were imaged using 5% of 488 nm laser for GFP::mec-12 and 1% of 561 nm laser for mCherry.For quantifying the ratio of the average intensities of  mCherry  and thei mCherry  worms co2 test (Fisher’s exact test) was used for comparing proportions, while ANOVA with a post hoc Tukey’s multiple comparisons test was used for comparing more than two groups. Bartlett’s test was conducted to test the homogeneity of variances before proceeding with ANOVA. The P-value, indicating the level of significance, was presented in each panel of the Figure to compare the respective groups. The sample number (n) for each experiment was mentioned in the respective Figure legend, along with the total number of biological replicates (N).The data analysis was performed using GraphPad Prism software version 9.0.2. The mean value and the standard error of the mean (SEM) were represented by the bar in the plots. The XS1 Figju1128 mutation. The 100-base pair ladder was used as a marker. In the gel picture, mutation showed linkage on two chromosomes, one on the third chromosome, which is because of klp-7(tm2143) (marked with red arrowhead) and another on the X chromosome (marked with red arrowhead). (B) Frequency plot of X-chromosome for mapping of the ju1128 mutation from the whole genome sequencing data using the method described by Minevich et al. (2012), with chromosome position (in megabases/Mb) plotted against the Normalized frequency of pure parental alleles. It peaks at the genomic position linked to the ju1128 mutation.(A) The Gel picture showing the result of Restriction Fragment Length Polymorphism (RFLP) mapping of (TIF)Click here for additional data file.S2 Figmbl-1(0) at the L4 stage. BDU neurons were visualized using Punc-86::GFP (kyIs262) and for visualization of touch neurons, Pmec-4::mCherry (tbIs222) and Punc-86::GFP (kyIs262) transgenes were used. The presence of physical contact between the PLM anterior neurite and BDU neuron is shown by the white arrowhead in the wild-type background which is lost in mbl-1(0) shown by the red arrowhead. (C) Quantification of the defect as shown in the image (A). N = 3 independent replicates, n (number of worms) = 25–32. (D-E) Representative confocal images (D) and schematics (E) of the gap junction synapse labeled with UNC-9::GFP(shrEx434) in the touch neurons, in the wild-type and mbl-1(0) backgrounds. The green arrows show the localization of UNC-9::GFP in the wild-type background, whereas in the mbl-1(0) the red arrow is pointed at the tip of PLM anterior neurite missing UNC-9::GFP localization. (F) Quantification of the percentage of defect shown in the image in panel D. N = 3–4 independent replicates, n (number of worms) = 30–35. (G-I) Representative confocal images (G) and illustration (H) of PLM neuron in the wild-type (muIs32) and mbl-1(tm1563); muIs32 backgrounds at A7 (seven-day adult) stage. White arrows are showing the visible gaps in the anterior neurite indicating degeneration. (I) Histogram showing the quantification of degeneration of PLM neurons at A3 (three-day adult stage), A5 (five-day adult stage), and A7 in wild-type and mbl-1(tm1563) background. N = 3 independent replicates, n (number of worms) = 16–26. For C, and F***P <0.001; Fisher’s exact test. ns, not significant.(A) Representative confocal images and (B) illustration of touch neurons (ALM and PLM) and BDU neurons in both wild-type and (TIF)Click here for additional data file.S3 Figmbl-1(0) (B) worms expressing a presynaptic reporter Pmec-7-ELKS-1::TagRFP (jsIs1075), shown in magenta color. The neuron is also labeled with diffusible reporter Pmec-7-GFP (muIs32), shown in green. The ectopic synapse in the PLM anterior process in the mbl-1(0) background is marked by a red arrowhead, whereas the original synapse is marked by a white arrowhead. (C) The histogram shows the percentage of PLM neurons with ectopic synapses in the mbl-1(0) background. N = 3 independent replicates, n (number of worms) = 25–30. (D-F) Representative images (D) and schematic (E) of D-type motor neurons labeled with Punc-25::GFP (juIs76) in the wild-type and mbl-1(0) at the L4 stage. Red arrow showing a defect in neurite growth in mbl-1(0) background. (F) The histogram shows the percentage of worms showing neurite defects in the mbl-1(0). N = 3–4 independent replicates, n (number of worms) = 30–50. (G) Representative confocal images of touch neurons in the mutants of different RNA binding proteins. The names of the mutants are mentioned next to the respective image panels. (H) Quantification of any defects in PLM morphology seen in the mutants mentioned in (G) N = 3 independent replicates, n (number of worms) = 80–130. For C, F, and H ***P <0.001; Fisher’s exact test. ns, not significant.(A-C). Confocal images of wild type (A) and (TIF)Click here for additional data file.S4 Figmec-7 (A), mec-12 (B), unc-76 (C), and sad-1 (D). (E and F) The histograms show the anterior (E) and posterior (F) gentle touch response index of the worm in the wild-type, mbl-1(0), mec-7(0), mec-12(0), mbl-1(0) mec-7(0) and mbl-1(0); mec-12(0) backgrounds. N = 3 independent replicates, n (number of worms) = 16–50. For E-F, **P < 0.01; ***P < 0.001. Error bars represent SEM. Statistical comparisons were done using ANOVA with Tukey’s multiple comparison test. ns, not significant.(A-D) Pictures depicting MBL-1/MBNL-1 preferential binding sequence and binding positions in the transcript of (TIF)Click here for additional data file.S5 Figtba-1, mec-7, mec-12, sad-1, and unc-76 genes. The sequence of these primers is given in the supplementary file sad-1 and unc-76 transcript in the wild-type and the mbl-1(0) background. (C) The histogram is showing the relative fold change of the transcript of mec-7, mec-12, sad-1, and unc-76 in the wild type and the mbl-1(0) backgrounds. These data were obtained from quantitative real-time PCR (qRT-PCR). Independent replicates (N) = 6 and the number of reaction (n) = 6–8. For C, ***P < 0.001; ANOVA with Tukey’s multiple comparison test. Error bars represent SEM. ns, not significant.(A) Illustration showing the positions of different primers, used for checking the transcript length or doing qRT-PCR, on (TIF)Click here for additional data file.S6 FigPmec-7::mec-7::gfp (cDNA) (shrEx473) (A-B), Pmec-4::gfp::mec-12 (cDNA) (shrEx492) (C-D), and Pmec-4::mCherry (tbIs222) (A-D) in the wild-type and mbl-1(0) backgrounds. The fluorescence intensity is quantified in an arbitrary unit from the anterior (Ant) and posterior (Pst) neurites of PLM, from 50 μm regions of interest (ROI) as shown in the mbl-1(0) backgrounds. For A-D, independent replicates (N) = 3–4 and the number of neurons (n) = 20–25. (E and G) Representative confocal images of the worms expressing Pmec-7::mec-7::gfp (genomic DNA) (shrEx474) and Pmec-4::mCherry (tbIs222) (E), and worms expressing Pmec-7::mec-7::gfp::SL2::mCherry (shrEx486) (G) in wild-type and mbl-1(0) background. (F and H) The histogram is showing quantification of the ratio (MEC-7::GFP/MCherry) of average fluorescent intensity from 50 μm regions of interest (ROI) as shown in Pmec-7::mec-7::gfp (genomic DNA) (shrEx474) and Pmec-4::mCherry (tbIs222) in arbitrary units (I) and histogram showing fluorescence intensities of Pmec-7::mec-7::gfp::SL2::mCherry (shrEx486) (J) in the wild-type and mbl-1(0) backgrounds. For F, H, I, and J, independent replicates (N) = 3–4 and the number of neurons (n) = 20–25. For A-D, F, H, and I-J, *P < 0.05; **P < 0.01; ***P <0.001; ANOVA with Tukey’s multiple comparison test. Error bars represent SEM. ns, not significant.(A-D) Histograms showing the absolute fluorescence intensities of (TIF)Click here for additional data file.S7 FigPmec-4::EBP-2::GFP (juIs338) in wild-type, mec-7(0), and mec-12(0) backgrounds. (B) Representative kymographs of EBP-2::GFP obtained in the wild-type, mec-7(0), and mec-12(0) backgrounds obtained from the above-mentioned ROIs. The green and magenta traces on kymographs represent the Plus-end-out (microtubule growth events away from the cell body) and Minus-end-out (towards the cell body) tracks, respectively. (C) The bar graph is showing the fraction of microtubules with plus-end-out’ (P) or ‘minus-end-out’ (M) polarity in wild-type, mec-7(0), and mec-12(0) backgrounds in the PLM anterior (Ant) and posterior (Pst) neurites. N = 3–5 independent replicates, n (number of worms) = 20–50. (D) The bar graph represents the number of EBP-2::GFP tracks (number of growing microtubules) in PLM anterior (Ant) and posterior (Pst) neurites in wild-type, mec-7(0), and mec-12(0). N = 3–5 independent replicates, n (number of worms) = 20–50. (E and F) Growth length (E) and growth duration (F) of the tracks, measured from net pixel shift in the X and Y axis, respectively, from kymographs shown in B. N = 3–5 independent replicates, n (number of tracks) = 1986–6064. For C-F, ***P < 0.001; ANOVA with Tukey’s multiple comparison test. Error bars represent SEM. ns, not significant.(A) Schematic of the PLM neuron showing the 30 μm Regions of interest (ROIs), marked in red and orange for anterior and posterior neurites, respectively. These ROIs were used for analyzing the time-lapse movies of (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file. 31 Oct 2022Dear Dr. Ghosh-Roy,C. elegans mechanosensory neuron through tubulin mRNAs' to PLOS Genetics.Thank you very much for submitting your Research Article entitled 'Musleblind-1 regulates microtubule cytoskeleton in mec-7 and mec-12 transcripts, how mbl-1 suppresses kpl-7, and what link exists between the regulation of mec-7/12 by mbl-1 and the microtubule polarity and dynamics.  Moreover, the manuscript will need to be structured within the context of previous published work, some of which has not been cited.  This includes the early work on neuronal development and polarization, as indicated by Reviewer 1, as well as the work on mbl-1 in synaptic formation(Shen lab) that should be presented as the entry point for this study of mbl-1 on synapses.  Finally, the manuscript will need to be carefully checked for grammatical errors, typos, and colloquial language, which populate the current version.  Based on these reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version. We cannot, of course, promise publication at that time.The manuscript was fully evaluated at the editorial level and by three independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript that need to be addressed to provide mechanistic insights, expand the data analysis, and strengthen the conclusions.  These are especially related, but not limited, to the insufficient data provided to support the conclusion that MBL-1 stabilizes the Should you decide to revise the manuscript for further consideration here, your revisions should address the specific points made by each reviewer. We will also require a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.plosgenetics@plos.org.If you decide to revise the manuscript for further consideration at PLOS Genetics, please aim to resubmit within the next 60 days, unless it will take extra time to address the concerns of the reviewers, in which case we would appreciate an expected resubmission date by email to Submission Checklist.If present, accompanying reviewer attachments are included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. 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All points should be backed up by data provided with the submission.Please be aware that our Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool.  PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.While revising your submission, please upload your figure files to the Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.PLOS has incorporated To resubmit, use the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.Yours sincerely,Massimo A. HilliardGuest EditorPLOS GeneticsGregory P. CopenhaverEditor-in-ChiefPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: The article by Puri et al. investigates the role of MBL-1 in neuronal structure and synapse localization in the mechanosensory neurons of C. elegans. It provides new insights into the importance of this molecule for correct neuronal structure. It also offers mechanistic insights by demonstrating interactions with mec-7, mec-12, and sad-1 mRNAS. Thus, overall it provides novel data on how an RNA binding protein interacts with cytoskeletal elements to mediate correct neuronal structure. As listed below, I have numerous suggestions for the authors to address before the manuscript can be considered for publication.Overall, the writing of the manuscript needs significant improvement. The grammar and sentence structure need to be improved to help with the readability of the manuscript, as there are many instances in which the meaning of the authors is unclear.It would be important to cite some of the early work on neuronal development and polarization in C. elegans in the introduction. For example, Adler et al., 2006 Nat Neuro; Chang et al., 2006 Curr Biol; Hilliard and Bargmann, 2006 Dev Cell; Prasad and Clark, 2006 Development; Quinn et al., 2008 Curr Biol.All gene names should be italicized.Figure 1.Panel A. The authors may wish to label the PVM neurons in their images to make them more understandable for those who are not familiar with the mec neurons of C. elegans.Panel C. Was the ALM defect analysed with a yes or no qualification, ie it either had a posterior neurite or it didn’t? This is not clear in the manuscript.Panel D and E. The stats are not clear in these two graphs – is the fosmid rescue line significantly different to the controls (ie WT and mbl-1(ju1128)?Is the fosmid able to rescue the tm1563 allele?Figure 2.Why did the authors choose to focus on the tm1563 allele for this figure (for images and rescue) when they primarily used the ju1128 allele in Figure 1?Panel A. In the image of the mbl-1 mutant, the ALM and PLM axons appear much fainter than the WT and possibly fragmented. Is there degeneration occurring in these neurons? Thus, is the short phenotype developmental or is it degenerative?Panel B. It’s interesting that the TRN-specific rescue is not as strong as the genomic or pan-neuronal rescues. Why do the authors think this is so? Is this difference significantly different?Panel F. The text states that “MBL-1::GFP was 238 highly enriched in many neurons including ALM and PLM touch neurons”. However, from the images provided it is impossible to see the PLM neurons. It appears that the authors have performed the required co-localization experiments using a mCherry marker, but this is not visible in these images. Please provide clearer and/or enlarged images and/or quantification to demonstrate that MBL-1 is expressed in the ALM and PLM neurons. Furthermore, do you find enrichment of MBL-1 in any areas of these neurons?Figure 3.The author may wish to consider separating this figure into two to make it easier to follow.Panel M/N. The functional decline in the posterior response is quite subtle. It would be good to show that this is rescued with the introduction of the rescue transgene to provide further strength the findings. Expanding subsequent findings, especially with mec-12, mec-7 and sad-7, to assess their effects on the mbl-1-mediated reductions in touch response would provide further strength to the proposed interactions and into their mechanisms of action.Figure 4.Both mec-7 and mec-12 mutants have previously been shown to present a high penetrance of axonal degeneration in the mec neurons. However, here the authors again refer to the phenotype as short neurites. The authors need to include additional experiments that clarify their phenotype as either developmental or degenerative.Figure 5.Panel D-E. I’m not sure that I follow the authors assumption that “This reduction observed in the mbl-1(0) mutants as compared to the wild type is interesting, as it illustrates the fate of the pre-existing transcripts in the absence of MBL-1 and in the absence of any new transcription. As transcription was blocked, the observed additional decrease could be attributed to the increased instability of the pre-existing transcripts in the absence of MBL-1.” It seems that the more plausible explanation would be that this experiment is showing the normal turn-over of the transcripts, as for both WT and mutant you show an ~40% decrease after treatment. Thus, the mutant is appearing to behave the same as the WT after treatment. As such, I am not convinced by the conclusion that “MBL-1 is regulating the stability of mec-7 and mec-12 mRNA in PLM neurons.”Panel F/G. Did the authors find similar results with mec-12?Figure 6.Panel B/C. These panels show interaction with mec-7, mec-12 and sad-1, but then mec-12 appears to be forgotten about for the next experiments in this figure and in the text, which only refers to mec-7 and sad-1. Why is this? Did the authors find that mec-12 did not interact with MBL-1 in the mec neurons in subsequent experiments? The experiments conducted on mec-7 and sad-1 in panels D-E should also be included for mec-12.Figure 7.Again, why wasn’t mec-12 included in these analyses? It is included in the model in panel F. Although MEC-12 and MEC-7 are most commonly known as the major subunits of microtubules in the mec neurons, there are several examples in the literature demonstrating that they can function quite differently to one another and influence specific phenotype in different ways or to different degrees. Thus, it cannot be assumed that they will behave the same if the experiments have not been performed.Panel F. The authors may wish to expand their model to make it more informative and reflective of their proposed mechanisms of action.Figure S2. The images are far too small to allow the reader to see the required detail. Yes, the schematics are of great assistance, but the original images need to be able to be seen.The text states “In the mbl-1(0) mutant, we noticed that on the dorsal side, there are oftengaps  indicating a synaptic defect”. However, the authors have only used a diffuse GFP marker to visualise these neurons. Further analysis using a synaptic marker in these neurons is needed to conclude that these gaps indicate a synaptic defect rather than shorter neurites (similar to the phenotype in ALM/PLM).Reviewer #2: Review_PGENETICS-D-22-01039Puri, Ghosh-Roy et al. described how C. elegans MBL-1/Muscleblind, an RNA-binding protein, promotes axon growth and synapse development by regulating microtubule polarity through controlling the level of tubulin mRNAs. They first identified mbl-1 as a suppressor for touch neuron defects in the klp-7 Kinesin mutant, followed by providing evidence that microtubule dynamics and levels of touch neuron-specific tubulins  are altered in the mbl-1 mutant. RNA-IP experiments showed that MBL-1 binds mec-7 RNA, suggesting that MBL-1 sustains MEC-7 levels through a posttranscriptional mechanism. Consistent with this notion, overexpression of mec-7 rescued the neurite defects of the touch neurons in the mbl-1 mutant. Most of the experiments in this study were reasonable, although some clearly need further refinement in terms of data analysis. However, it remains unexplored how regulation of tubulins by MBL-1 is linked to microtubule polarity and dynamics in the touch neurons. The interaction between mbl-1 and sad-1 is not explored at a deeper level, making one wonder whether the molecular mechanisms by which mbl-1 regulates tubulins and sad-1 are similar or distinct. Such unaddressed issues compromise the mechanistic depth of the paper.Major points:1. The paper needs a thorough editing check to correct the numerous grammatical errors and typos in writing. The authors should be more careful in checking their submitted manuscript. In addition, the authors have a tendency of using terms that are either too casual, or seem alienated to the audience without explanation. For example, in line 311 and line 314, “mode value” and “fraction polarity value” were used to describe the polarity defects of the mbl-1 mutant. I suggest the authors revise all terms like these to make their paper better received by the audience.2. The suppression of klp-7 mutant phenotypes by mbl-1 remains unexplained. The authors need to test the following: (1) Whether microtubule dynamics in the klp-7 are different from the WT and the mbl-1 mutant, and whether defects of microtubule dynamics in the klp-7 mutant are suppressed by the mbl-1 mutation; (2) Whether mec-7 and mec-12 levels are altered by the klp-7 mutation, and whether these defects are again corrected by adding the mbl-1 mutation.3. What is the link between mec-7/mec-12 regulation by mbl-1 and intact microtubule polarity and dynamics in the touch neurons? As had been stated above, this is the most important mechanistic issue for the current study that needs to be rigorously explored by experiments.4. What is the basis of axon defects in the D-type motor neurons of the mbl-1 mutant? Obviously, this is unlikely to be the consequence of altered mec-7 and mec-12 levels, as these two tubulin genes are not expressed in DD or VD neurons. Can the authors speculate from the candidate MBL-1 bound RNAs  and elaborate on this issue (by discussion or experiments)?5. Figure 5D, 5E: The interpretation is flawed that instability of mec-7 and mec-12 transcripts is increased in the mbl-1 mutant. Since the expression of these two genes is already low in the mbl-1 mutant, the authors need to normalize the levels of mec-7 and mec-12 transcripts after Actinomycin D to those at baseline, and compare this value to that of the wild type animals. Without such quantification, the current conclusion that mbl-1 promotes the stability of mec-7 and mec-12 transcripts is premature and unsupported.6. Figure 5F, 5G, Line 467-472: I could not find statements of using Actinomycin D in this experiment, so I assumed that it was conducted without Actinomycin D. If so, then the data presented here cannot be used to support the role of MBL-1 in maintaining MEC-7 stability. Moreover, the cytosolic mCherry protein was expressed from a different transgene using a different promoter (Pmec-4) from the mec-7-expressing transgene driven from the mec-7 promoter. As such, comparing the GFP to mCherry ratio using these two transgenes is not a valid way of normalizing MEC-7::GFP quantity. The authors need to create a Pmec-7::MEC-7::GFP::SL2::mCherry transgene for this purpose. That said, these experiments only explore the role of MBL-1 in controlling MEC-7 level without specifically examining its function in promoting MEC-7 stability, which clearly requires the use of Actinomycin D. These experiments need rigorous revision.7. Figure 6: A negative control for the RNA-IP is necessary to show that MBL-1 does not bind most RNAs in a non-specific fashion. Using the KLP-7::GFP as a control is fine, but the authors need to show that not all RNAs are enriched in the RIP samples of MBL-1::GFP worms.Minor points:1. Line 47 and 107: please explain sad-1 (such as “sad-1/SAD BRSK kinase”)2. Figure 1B: It is better to just show the gene structure of mbl-1 and what the ju1128 and tm1563 alleles are, rather than showing the fosmid contents.3. The authors are suggested to provide high power images for the neurite and synaptic branch defects of the mbl-1 mutants as well as MBL-1::GFP in Figure 2. It is hard to see the details of the ALM and PLM phenotypes in the current images .4. Line 122: The term “multiple axon-like phenotype” (of ALM) is a bit confusing. Although there is still debate whether this is a bipolar ALM phenotype or a misguidance defect of the ALM process, the term that the authors used does not help clarify the issue. I suggest that they just say “ectopic ALM process” .5. Line 182: Please correct this sentence: “making a ventral synaptic branch the PLM axon makes a synapse…”6. Line 192: “Pmec-4” should be italicized7. Line 242: Gaps in the dorsal axons of D-type motor neurons should be interpreted as defects in axon extension similar to the short ALM and PLM neurites, rather than as synaptic defects. The authors should examine the distribution of RAB-3 and ELKS-1 in the D-type motor neurons of the mbl-1 mutant to substantiate their conclusion that mbl-1 is required for synaptic development in this class of neurons.8. Line 246-247: Please annotate these genes succinctly.9. Line 252: correct “caused due”10. Figure 3: The green and purple lines in embedded in Figure 3B are very hard to see. Figure 3J: Some lines are skewed.11. Line 263: “%” should be “percentage”.12. Line 268: “smaller” should be “shorter”.13. It is not clear what microtubule “arrangement” means in the paper. Microtubule “polarity” or “orientation” could be a better term, or the authors need to provide convincing justification for using such a term.14. Line 382-387: I am confused here as the authors said that they found mec-7 and mec-12 were among genes found to be downregulated by mbl-1 in some published reports but claimed that they could not find genes in this group that were related to GO terms of axon development or synapse structure. Aren’t mec-7 and mec-12 two such genes?15. Line 420: The expression of “0.4717 +/- 0.03720 fold” is very strange and inappropriate. This could be seen again in Line 456. The authors should carefully revise their use of scientific expression. As has been stated above, this manuscript needs a thorough editing of writing. The senior author should take the responsibility to ensure the quality of writing.16. Figure 6F, left: The protein band for MBL-1::GFP seems to be absent in the total lysate of the MBL-1::GFP::FLAG animals. The authors need to exlain this. A minor point is that the protein bands in the total lysates of the MBL-1::GFP::FLAG and Pmec-4::MBL-1::GFP look quite different. Why?17. Figure 7A, the third image from top: That should be the AVM neuron, not the AVA neuron.Reviewer #3: Puri et al. reveal the role of mbl-1 in neurite and synapse biology through regulation of microtubule cytoskeleton components. In a genetic screen for suppressors of ectopically extended neurites in the ALM neuron in C. elegans, they identify a mutation in mbl-1 that shortens neurites in klp-7 mutants that have enhanced microtubule stability. mbl-1 mutants displayed mixed microtubule polarity and defects in axonal transport of cargo. MBL-1 is an RNA-binding protein, and the authors show evidence that it regulates the mRNAs of the microtubule genes mec-7 and mec-12, as well as sad-1. The authors propose that MBL-1 binds these mRNAs and stabilises them or mediates splicing in the case of sad-1. Overall, the manuscript is technically robust, novel, and interesting, however some of the mechanistic data into MBL-1 function needs improving as does the explanation of results that do not fit the conclusions made. There are some outstanding questions that also remain unaddressed.Major:1. The interpretation that MBL-1 stabilises the mRNA molecules of mec-7 and mec-12 is based on insufficient data. While the use of Actinomycin D is good, it may not block transcription fully and the authors need to prove whether this is indeed the case in their animals. Moreover, the translational MEC-7::GFP reporter cannot distinguish between transcriptional or post-transcriptional mec-7 regulation. Therefore, other orthogonal evidence is required to conclude that MBL-1 stabilises these specific transcripts.2. Some of the data do not fit the conclusions. From the RIP experiments, MBL-1 associates with mec-7 and sad-1 mRNAs in mechanosensory neurons, but not mec-12. In light of this, how do you reconcile its regulation of mec-12 in PLM? Sad-1 mRNA levels or splicing are not altered in mbl-1(0) mutants, yet it associates with MBL-1 protein. If association results in mRNA stability or splicing, why aren’t sad-1 mRNA levels lower or altered in length in animals lacking mbl-1?3. There are also some unanswered questions. Why do PLM anterior processes develop a mixed microtubule polarity if mec-1 and mec-12 mRNA levels are reduced in mbl-1(0) mutants? Why is the number of growing microtubules increased in mbl-1(0) mutants?MINOR.At times, the manuscript is written in a descriptive manner (phenotypic data) and could be improved with more contextualisation and/or insight into the functional consequences of the observed neurite changes. Highlighting the conceptual advances or the point of each result throughout the manuscript may also improve readability in some sections.Figure 2B: Authors should clarify in the main text and legend that TRN refers to mechanosensory neurons under pmec-4.Page 8 – line 183 – only photos for one of the mutant alleles is shown in fig. 2A.Page 8 – line 198 – The UNC-9::GFP reporter appears as puncta throughout the body of the wild-type animal in fig. S2D. Many of these puncta are missing in the mbl-1(0) mutant. Can the authors explain this? In addition, it is also very difficult to understand how the schematics in panel E of this figure were derived from the representative images. For example, there are many UNC-9::GFP puncta along the wild-type PLM axon that are not included in the schematic. What are these? This result needs better clarification.Page 15 – line 351 – According to Table S3, 22 genes are noted are assessed, rather than 13.Page 21 –line 469 - missing mcherry transgene nomenclature.Page 15 -lines 342 – 362 should be condensed. It reads as repetitious.Figure 4 - Authors should indicate what fluorescent reporter is used for the imaging and analysis. Figure needs images of controls/wild types for reference.Some grammatical and sentence structure errors. Some parts of the text could be simplified to avoid confusion. The protein of interest is spelt incorrectly in the title.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.archive. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License. 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Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.PLOS has incorporated To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.Please let us know if you have any questions while making these revisions.Yours sincerely,Massimo A. HilliardGuest EditorPLOS GeneticsGregory P. CopenhaverEditor-in-ChiefPLOS GeneticsDear Dr. Ghosh Roy,Thank you for submitting a revised version of your manuscript. The reviewers have examined the paper and they have found it highly improved compared to the original submission. However, they found that there are still some editing elements that needs to be addressed before the paper can proceed further. Importantly, they also highlighted that the manuscript still needs improvement in the writing, grammar, and sentences structure; please ensure that these also are addressed before your final submission.Unless you believe additional time is needed, I trust a revised version can be submitted within 2 weeks.I look forward to receive your revised manuscript.Thank you,Kind regards,Massimo.Reviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: The authors have addressed most of my previous concerns and the paper is now much improved from the first submission. However, I still have some minor concerns that should be addressed prior to the manuscript being published.1. The writing still needs significant improvement for clarity, and to correct grammatical errors and issues with sentence structure.2. The title should be revised for clarity and to better describe the study. I suggest the following: "Muscleblind-1 interacts with tubulin mRNAs to regulate the microtubule cytoskeleton in the C. elegans mechanosensory neurons"3. The authors should provide a definition for the sad-1 gene in the abstract. Currently, no indication is given for what this gene is, and therefore there is no capacity for the reader to grasp the importance of the interaction between MBL-1 and sad-1.4. The TRN phenotypes of the mbl-1 mutants are remarkably similar to those observed in the absence of MEC-17 . Yet, there is no mention of MEC-17 in the manuscript. The authors should at least include a mention of the similarities and potential involvement of MEC-17 in their discussion.Reviewer #2: The authors had done significant amount of work for the revision, and these additional data strengthened the conclusion and clarified most of the issues that I raised during the initial review of the paper. The revised manuscript was much better than the original one. I think the paper can now be considered for publication at PLoS Genetics, after the following minor points are addressed:1. sad-1 should be explained in the abstract.2. In Author summary, the last statement "... might help mitigate the diseases such as muscular dystrophy caused due to misregulation of MBNL in humans" is better removed or tuned down. The study was conducted in neuronal cells, which have very different gene expression patterns and cell biology to those of the muscle cells. The current statement is misleading and exaggerating.3. Line 119: The anterior cells should include AVM? Or the authors should just remove "anterior cells" and "posterior cells" to avoid confusion. As my comments in the initial review, the authors should avoid using jargons and instead stick with the standard terminology of the worm community.4. I cannot find description of the suppressor screen in the Materials & Methods section. A few descriptions will be necessary, such as the mutagenesis method (EMS?), an estimation of the screen size (number of haploid genomes screened), etc. By contrast, a detailed description of mapping ju1128 in the Results may not be necessary or should be streamlined (line 136-147).5. Line 175: "mbl" should be italicized.6. Line 205: It should read "In both mutant alleles of mbl-1"7. The y-axis scales of figure S3C and S3H should be mended as a full 100% scale.8. Figure S7E: Statistics should be carefully checked again, as the data of the WT and mec-7 or mec-12 mutants look quite similar at the first glance.Reviewer #3: Puri et al have submitted a revised version of their manuscript. They have addressed all of my comments well and the manuscript is an improvement overall.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  have corrected several grammatical errors/typos.  Please incorporate those corrections as you prepare your final draft for the production team .  Congratulations on the acceptance of your paper, and thank you for choosing PLOS Genetics as a venue for your work!Before your submission can be formally accepted and sent to production you will also need to complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. 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Mycobacterium tuberculosis (Mtb). It is also known for its synergistic effects in combination with pyrazinamide (PYR) and moxifloxacin (MOX). Fixed-dose combination powder formulations of either PYR and PA or PYR and MOX were prepared for inhaled delivery to the deep lung regions where the Mtb habitats were located. Powder formulations were prepared by spray drying using L-leucine as the aerosolization enhancer and were characterized by their particle size, morphology and solid-state properties. In vitro aerosolization behaviour was studied using a Next Generation Impactor, and stability was assessed after storage at room temperature and 30% relative humidity for three months. Spray drying with L-leucine resulted in spherical dimpled particles, 1.9 and 2.4 µm in size for PYR-PA and PYR-MOX combinations, respectively. The powder formulations had an emitted dose of >83% and a fine particle fraction of >65%. PA and MOX showed better stability in the combination powders compared to PYR. Combination powder formulations with high aerosolization efficiency for direct delivery to the lungs were developed in this study for use in the treatment of latent and multidrug-resistant TB infections.Tuberculosis (TB) is an infectious disease resulting in millions of deaths annually worldwide. TB treatment is challenging due to a huge number of global latent infections and due to multidrug-resistant forms of TB. Inhaled administration of anti-TB drugs using dry powder inhalers has various advantages over oral administration due to its direct drug delivery and minimization of systemic side effects. Pretomanid  is a relatively new drug with potent activity against both active and latent forms of  Mycobacterium tuberculosis (Mtb), and it was one of the leading causes of death worldwide due to a single microorganism until 2021 [Tuberculosis (TB) is a disease mainly of the lungs caused by til 2021 . Around til 2021 ,5. Althotil 2021 . Growingtil 2021 ,8,9.The currently recommended treatment regimen for drug-susceptible TB includes oral treatment with first-line agents for a two-month ‘intensive phase’, followed by a four-month ‘continuation phase’. In the initial intensive phase, the drugs rifampicin, isoniazid, pyrazinamide and ethambutol are used in combination for treatment . In spitTB patients who do not respond to the first-line treatment are classified as having drug-resistant TB, which can be either multidrug-resistant TB (MDR-TB) or extensively drug-resistant TB (XDR-TB). In MDR-TB, the mycobacteria are resistant to at least isoniazid and rifampicin, the two potent first-line anti-TB agents. In some cases, more severe drug resistance can develop as XDR-TB, which is an aggravation of MDR-TB with further resistance to the fluoroquinolones and at least one of the injectable second-line drugs—capreomycin, kanamycin and amikacin. Due to the huge economic and public health burden of drug-resistant TB, there is an urgent need for new and potent treatment strategies that efficiently kill the mycobacteria and shorten the duration of treatment . MDR-TB-Currently, the use of fixed-dose combination tablets is recommended over separate drug formulations in the treatment of patients with drug-susceptible TB . A fixedInhaled delivery of anti-TB drugs as dry powder inhalers (DPIs) is a safe, reproducible, non-invasive and convenient approach to delivering high concentrations of drugs to the lungs as well as systemic circulation. This route of drug administration can also achieve high bioavailability due to avoidance of the first-pass metabolism, can reduce the side effects that result from excessive systemic exposure to the drugs and can prevent the potential drug resistance build-up ,19,20. D® Milli-Q® Reagent Water System  filtered through 0.45 μm membrane filter was used throughout the study.The materials used in this study include the following: pyrazinamide ; PA-824 ; moxifloxacin hydrochloride ; L-leucine ; silicone oil ; and size-3 hard gelatine PEG capsules . All other chemicals and solvents used were of high-performance liquid chromatography (HPLC) grade and purchased from Merck, Germany. Freshly collected ultrapure water , and those for PYR-MOX-LEU formulation were prepared by dissolving supplied PYR, MOX and LEU at 64, 16 and 20% w/w, respectively, in ethanol and water (70:30% v/v). The mass ratio of PYR:PA was 8:1, which was selected based on the dose used in the phase II study conducted by Dawson et al. in 2015 [w/v final concentration and sonicated for 30 min prior to spray drying. Similarly, in the case of PYR-MOX-LEU formulation, the feed solution of 0.5% w/v final concentration was obtained by mixing the solutions containing 320 mg PYR in 70 mL ethanol and those containing 80 mg MOX and 100 mg LEU in 30 mL ultrapure water.Two powder formulations were prepared with a fixed-dose combination of either PYR and PA with LEU or PYR and MOX with LEU. Feed solutions for the PYR-PA-LEU formulation were prepared by dissolving supplied PYR, PA and LEU at 71.1, 8.9 and 20%  in 2015 . For theA BUCHI B-290 Mini Spray Dryer  with a high-performance cyclone connected to the B-295 Inert Loop in a closed mode was utilized to prepare spray-dried powder formulations. High-purity dry nitrogen gas was used as the atomizing drying gas. The stainless-steel inner nozzle tip diameter was 0.7 mm. The conditions for spray drying included an inlet temperature of 70 °C, 50% aspirator control, a feed flow rate of 2 mL/min and a spray flow rate of 819 L/h (height 65 mm). Under these conditions, the outlet temperature varied between 41 and 46 °C. The spray-dried powders were then collected and stored in screw-capped glass vials. These glass vials were protected from light and stored in a desiccator at room temperature (22 ± 3 °C) and at a relative humidity of 30 ± 2% until analysis.The percentage yield (% yield) of the prepared powders was estimated by dividing the collected powder weight by the total weight of the solids used for preparation of feed solution and expressed as a percentage.w/w/w) and PYR-MOX-LEU (64:16:20% w/w/w) were prepared by simple mixing of accurately weighed quantities of respective drugs and LEU using a spatula for 5 min.Physical mixtures of PYR-PA-LEU  at a speed of 200 rpm for 30 min. The mixtures were then sonicated using a bath sonicator  for 15 min and agitated by a vortex mixer to fully dissolve the powder. The samples were then filtered through a 0.22 µm membrane filter, and the concentration of each active ingredient in the samples was measured using HPLC analysis.For determination of active ingredients  in the spray-dried powder formulations, 5 mg of each powder sample (n = 3) was dissolved in 100 mL of 20:80% A field emission scanning electron microscope  was used to examine the surface morphology of powder samples. Each sample was mounted on a metal stub with a double-sided adhesive carbon tape by light dusting method. Prior to SEM imaging, the powder samples were coated with a thin layer of gold alloy (10 nm) in vacuum using the sputter Emitech K575X high-resolution sputter coater . The SEM micrographs were then obtained at various magnifications  with an accelerating voltage of 5 kV.https://imagej.net/ij/index.html accessed on 10 July 2023). Representative SEM images of each powder sample at various magnifications were analysed by measuring the diameter of a minimum of 300 particles.The geometric mean particle diameter of the spray-dried powder was determined from the SEM images using ImageJ 1.48 software  calibrated with indium . Approximately 5 mg of the powder sample was weighed into a standard flat-bottomed aluminium pan (TA instruments), and the exact weight of each sample was recorded. The pan loaded with sample was then hermetically sealed with the standard aluminium lid. All DSC samples were heated from 30 °C to 300 °C at a heating scan rate of 5 °C/min with nitrogen gas purge rate of 50 mL/min. For blank measurement and comparison, an empty aluminium pan pressed similarly to the sample pans was used.Thermogravimetric analysis of the supplied materials and spray-dried powder formulations was performed through use of a TGA Q50 Thermogravimetric Analyser . The platinum sample pan  was loaded with approximately 5–10 mg of the powder sample and heated under a nitrogen purge (40 mL/min) from 30 °C to 300 °C at a rate of 5 °C/min. The percentage weight loss during the heating process was analysed using Universal Analysis 2000 software v. 4.1D .Thermo-microscopic changes of the supplied material and spray-dried powder formulations were recorded using a phase-contrast light microscope  equipped with a polarizer, as well as a hot stage  with a Mettler Toledo FP90 central processor. A microscopic glass slide with a coverslip containing the sample was heated from 30 °C to 300 °C at a rate of 5 °C/min. The heating program was controlled using Image Pro Plus Software Version 2.7 , and images were digitally captured via an OPTIKAM PRO5 digital camera .To investigate the crystallinity of the powder samples, analysis was carried out using an X-ray diffractometer  equipped with a rapid real-time multistrip X’Celerator detector. Each sample was prepared by loading and compressing the powder in a flat plate geometry. Then, the sample was placed in the diffractometer for scanning over an angular range of 5–35° (2θ). Under ambient room conditions, the Cu Kα radiation operated at a generator power of 40 kV and current of 30 mA. The results were analysed by the PANalytical High Score software (version 4.0). TM, PIKE Technologies, Madison, WI, USA) was used to obtain ATR-FTIR spectra of the supplied materials and spray-dried powder formulations. The experimental procedure involved sample placement onto the ATR diamond crystal, followed by scanning (64 scans at 4 cm−1 resolution) over the wave number range of 4000–400 cm−1.A Varian 3100 FTIR  equipped with an attenuated total reflectance accessory (GladiATRv/v water: methanol). The collected samples were then made up to a final volume of 100 mL , 25 mL  and 20 mL (stage 2–7). The amount of drug in each was quantified by HPLC.The aerosol dispersion performance of spray-dried powder formulations was assessed utilizing a Next Generation Impactor  equipped with a Copley HCP5 vacuum pump and a Copley TPK 2000 critical flow controller. To prevent particle bouncing, all stainless-steel NGI cups were pre-coated with 15–30 µL of silicone oil per cup. Each size-3 hard gelatine PEG capsule  was filled with 20 mg of powdered sample. Aerosolization of the encapsulated powder was achieved using the Foradil Aerolizer . Before beginning aerosolization, the air flow rate was verified and adjusted to 100 L/min using a Copley DFM 2000 flow meter in conjunction with an electronic digital flow meter . The liberated powder from each capsule was drawn through the NGI at a flow rate of 100 L/min for 2.4 s. At this flow rate, the cut-off diameters are 5.27, 2.40 and 1.32 mm at stages 3, 4 and 5, respectively [Aerodynamic parameters, including emitted dose (ED) and fine particle fraction (FPF), were calculated as reported by Eedara et al. (2016) . ED repr18) column  preceded by a C18 security guard column . The samples (20 µL) were analysed in the isocratic mode using the following analytical conditions. For analysis, the mobile phase was composed of 30 mM orthophosphoric acid (pH 2.5 adjusted with triethylamine) and methanol (55:45% v/v), and the flow rate was 1 mL/min. The detection wavelengths for PYR, MOX and PA were 269 nm, 296 nm and 330 nm, while retention times were 3.5 min, 7.8 min and 17.7 min, respectively. The method was validated prior to use, and the accuracy and precision were within the specified limits of ±15% and <15%, respectively.The HPLC procedures were fully validated prior to their routine use . A Shimaw/v polyethylene oxide 5000 kDa and 0.4% w/v Curosurf® in PBS) spread uniformly on it. The dissolved fractions of the drug which permeated through the dialysis membrane were collected via an outlet at the predetermined time points of 2, 4, 6, 8, 10, 15, 20, 25, 30, 45, 60, 90, 120, 180 and 240 min and the samples were analysed by HPLC for quantification of the drugs.The in vitro dissolution study of PYR-PA-LEU and PYR-MOX-LEU powder samples was carried out using a custom-made dissolution apparatus, as reported previously ,26. The All spray-dried powder formulations were stored in airtight screw-capped glass vials at 30 ± 2% relative humidity under a desiccator at room temperature and were protected from light. The powder samples were tested at 0 and 3 months for the drug content, in vitro aerosolization, surface morphology and solid-state properties following the same methods mentioned above. p values less than 0.05 were considered statistically significant.All data were processed and represented as mean ± standard deviation values. Statistical analysis for comparison of groups was carried out by one-way analysis of variance (ANOVA) followed by a Student–Newman–Keuls post hoc test using GraphPad Prism 5 software . The percentage yield for each formulation was calculated, and the results were summarized in SEM was used to visualize the particle shape and size, which are the determinants of the aerosolization performance of dry powder formulations. The SEM images of the supplied materials and the spray-dried powder formulations are represented in The spray-dried powder formulations PYR-PA-LEU and PYR-MOX-LEU E,F appeaParticle size analysis was carried out for the spray-dried powder formulations (PYR-PA-LEU and PYR-MOX-LEU) using the SEM images. As shown in The thermal characteristics of the supplied materials and the spray-dried powder formulations were studied using DSC, and the thermograms are shown in DSC thermogram of PYR-PA-LEU physical mixture E showed The spray-dried powder formulation, PYR-PA-LEU G, exhibiThe TGA curves shown in The PYR-PA-LEU formulation showed a weight loss from 100 °C corresponding to the loss of PA. From 150 °C, the rapid rate of weight loss is attributed to both PA and PYR. From 250 °C, the weight of LEU started to decrease. At the end of the experiment, almost 95% of the total weight was lost, and the remaining was the weight of half of the PA amount. Similarly, in PYR-MOX-LEU, the rapid rate of weight loss was due to PYR and MOX. However, the rate was slower compared to that in PYR-PA-LEU due to a slower loss of MOX. The remaining weight was also higher because of a higher amount of MOX in the formulation.The hot-stage microscopy was used to visualize the change in powders with respect to the increase in temperature ranging from 30 to 300 °C. Supplied PYR A exhibitThe spray-dried powder formulations appeared to be dark, fine powders, indicating their amorphous nature after the spray-drying process. Particles of PYR-PA-LEU powder E startedXRPD analysis was conducted for powder samples to study the crystallinity of each sample and its polymorphic form before and after spray drying. XRPD is important to understand whether the powder samples are crystalline or amorphous. Amorphous particles are thermodynamically unstable and tend to recrystallize, and changes in such properties ultimately lead to unexpected effects on the aerosolization performance of the powder formulations.The spray-dried powder formulations of PYR-PA-LEU and PYR-MOX-LEU G,H showe−1, 3287 cm−1 and 3146 cm−1 in the FTIR spectrum , which is due to the smaller aerodynamic diameter of the PYR-MOX-LEU powder formulation as indicated by its MMAD values. Thus, smaller powder particles are more likely to be delivered into the deep lungs. The in vitro aerosolization performance of both PYR-PA-LEU and PYR-MOX-LEU formulations was similar when analysed using NGI . Both pow/v PEO in PBS) with lung surfactant, LS (0.4% w/v Curosurf), at 0.4 mL min−1 perfusate flow rate . Both spray-dried formulations (PYR-PA-LEU and PYR-MOX-LEU) showed faster permeation of PYR with >75% PYR permeation in <30 min. This indicates rapid dissolution of water-soluble, low molecular weight (123.1 g/mol) PYR from the respirable composite dry powder particles compared to the less soluble, high molecular weight PA (359.2 g/mol) and MOX (401.4 g/mol). PYR, from a spray-dried large porous particle powder formulation, is reported to be rapidly absorbed into the systemic circulation after intra-tracheal insufflation in laboratory rats with an average tmax (time to maximum plasma concentration) of 11.4 min [11.4 min . These o11.4 min .p < 0.05). MOX had similar aerodynamic parameters, which indicate its aerosolization stability over 3 months. Upon comparison of these aerosolization stability results, we can observe that PA might have helped protect PYR from degradation. However, this is contrary to the results from SEM analysis of stored samples (in the following discussion), which showed the formation of solid bridging between particles. Further study is necessary to investigate the mechanism behind this observation.The drug content of both formulations was within 90–100%, like fresh samples, which indicates the chemical stability of prepared spray-dried formulations for the short term. Due to the variation in the stability of the formulations, an XRPD analysis was also performed, the results of which are shown in High-dose DPI formulations containing either PYR and PA-824 or PYR and MOX were developed in this study using a spray-drying technique. To ensure that high doses of drugs can be delivered to the deep lungs in order to target the macrophages containing the mycobacteria, L-leucine was used as an aerosolization enhancer. The powder particles spray-dried with L-leucine typically exhibited porous, spherical morphology with a corrugated surface and size less than 5 μm in diameter. The rough surface of the powders helped reduce the inter-particulate forces, improved powder dispersion and aerosolization and facilitated the delivery of the powders to the deep lungs. In addition, incorporation of L-leucine in the powder formulations resulted in a polymorphic change of pyrazinamide from α-form to γ-form. The stability study conducted for three months for the PYR-PA-LEU and PYR-MOX-LEU formulations suggested that PA-824 and MOX within those combinations showed good stability; however, pyrazinamide had a significantly lower FPF at 3 months. This observation suggested the need for further investigation to improve the stability of pyrazinamide in fixed-dose combination powders for inhalation.


Hepatitis D virus (HDV) is classified into 8 genotypes (1 to 8) and several subgenotypes. In Brazil, HDV-3 and HDV-1 predominate; however, most of the diagnosis efforts and molecular studies are directed to the area of endemicity of the Amazon Basin. Here, we determined the molecular epidemiological profile of circulating HDV in Brazilian HBsAg-positive patients between 2013 and 2015 in areas of endemicity and non-areas of endemicity. From 38 anti-HDV-positive individuals, 13 (34.2%) had detectable HDV-RNA and 11 (28.9%) were successfully sequenced. Partial HDAg (~320 nt) sequencing followed by phylogenetic analysis with reference sequences resulted in the identification of HDV-3 , HDV-5 , and HDV-8 . Most HDV-3 samples  were found in the endemic North region, while one was found in Central-West Brazil, a non-area of endemicity. HDV-5 and 8, genotypes native from African countries, were found in São Paulo, a cosmopolitan city from Southeast Brazil with a high circulation of immigrants. Phylogenetic analysis of HDV-8 strains indicated that the sample determined in our study, along with previously reported sequences from Brazil, formed a highly supported monophyletic clade, likely representing a putative novel HDV-8 subgenotype.IMPORTANCE Considered a neglected pathogen until the last 2 decades, an increase in the availability of genetic data of hepatitis D virus (HDV) strains around the world has been noticed recently, resulting in the proposition of different classifications. Our study aimed to determine the molecular epidemiological profile of HDV isolates circulating in areas of endemicity and non-areas of endemicity in Brazil. Based on the analyzed fragment, HDV-8 sequences clustered out of the clades formed by subgenotypes 8a and 8b might suggest the identification of a novel subgenotype, putatively designated subgenotype 8c. Our findings demonstrate the importance of continuous epidemiological surveillance to map HDV spread pathways and the introduction of imported variants. It also reinforces that as the amount of HDV genomes generated and reported increases, we will have changes in viral classification and, consequently, in our understanding of the dynamics of variability of this viral agent. Hepatitis D virus (HDV) is a defective virus that requires hepatitis B virus (HBV) to replicate. Thus, hepatitis D is caused by the simultaneous infection of HBV and HDV (coinfection), or by secondary infection by HDV in HBV carriers (superinfection). HBV/HDV coinfection is the most severe form of viral hepatitis, often accelerating progression to cirrhosis and hepatocellular carcinoma .It is estimated that 5% of HBV carriers are infected with HDV, resulting in 12 to 60 million seropositive individuals worldwide . HDV endConsidered a neglected pathogen until the last 2 decades, an increase in the availability of genetic data of HDV strains around the world has been noticed recently, resulting in the proposition of different classifications for HDV –10. Base–Most studies conducted in Brazil are focused on areas of endemicity. However, a global understanding on HDV genetic variability is important to reach the World Health Organization (WHO) goal to eliminate viral hepatitis as a public health threat by 2030. This study aimed to determine the molecular epidemiological profile of HDV isolates circulating in areas of endemicity and non-areas of endemicity in Brazil.Of 38 anti-HDV positive samples from a previous survey  that werPhylogenetic analysis indicated the circulation of HDV-3 in 9/11 (81.8%) samples, eight from the endemic North Region where the Amazon Basin is located, and one from the state of Mato Grosso do Sul, located in the Central-West region (non-area of endemicity), which borders the Amazon Basin. The other two samples were obtained from individuals living in the state of São Paulo, Southeast region (non-area of endemicity), and were classified as HDV-5  and HDV-8  .Intragenotype genetic distance within HDV-3 sequences considering the 320-nt fragment analyzed here was 0.016 ± 0.003. This high genetic similarity of HDV-3 sequences prevented the formation of well-defined clusters distinguishing subgenotypes 3a, 3b, and 3c.Phylogenetic analysis performed on a genotype-specific data set composed of HDV-5 sequences indicated that the HDV-5 sequence characterized here clustered with sequences classified as subgenotype 5b, presenting high similarity with a sequence from Guiné-Bissau (tree not shown). The genetic distance between the group of sequences characterized as HDV-5a and HDV-5b was 0.085 ± 0.011 while the intragroup distance was 0.064 ± 0.009 for HDV-5a sequences and 0.059 ± 0.009 for HDV-5b.l-HDAg analyzed and the comparison of deduced amino acid sequences of HDV subgenotypes 8a, 8b, and the putative subgenotype 8c.Regarding HDV-8, our sequence was closely related to a sequence from Namibia and clustered in a distinct monophyletic clade together with Brazilian sequences from Maranhão, a state located in the Northeast region of Brazil , 16 Fig. Based oThis study describes the circulation of three HDV genotypes  in Brazil, in both areas of endemicity (North region) and non-areas of endemicity  . Surveillance of hepatitis D has been restricted to chronic HBV carriers living in the North Region, with few reports from states outside the Amazon Basin. Nevertheless, knowing the scope of HDV infection in Brazil and accessing the genetic diversity of this virus inside and outside regions of endemicity can provide useful information to contextualize Brazil in face of the goal proposed by WHO to eliminate viral hepatitis by 2030. Furthermore, HDV molecular characterization is a useful tool for detecting the introduction and circulation of imported genotypes as well as the most plausible routes of transmission throughout the country.Previous reports have shown that HDV-3 is the most prevalent genotype in Brazil, with a minor presence of HDV-1 , 15–18. 15–Here, no HDV-1 was found among the 11 HDV-RNA positive samples. HDV-3 was described in 9/11 (81.8%) samples, all but one from individuals living in the endemic North region. The remaining HDV-3 isolate was from an individual from the state of Mato Grosso do Sul, Central-West region. Even considered a non-region of endemicity, Central-West borders four states in the North region, which makes viral dissemination more feasible.Despite HDV-3 being the most divergent of all HDV genotypes, it presents a restricted intragroup genetic variability , 10. ConGenotypes HDV-5 and HDV-8, usually found in Africa , 21, 22,HDV-5 has been previously reported in Cameroon, Mauritania, and Nigeria . Howeverth to the mid-19th century. In our study, analysis of partial HDV-8 genome demonstrated the existence of a highly supported separated monophyletic clade, composed by other Brazilian HDV-8 sequences and a sequence from Namibia, apart from subgenotypes 8a and 8b. The genetic distance between sequences from this clade and HDV-8a or HDV-8b sequences was higher than that observed between subgenotypes 8a and 8b (0.14 versus 0.08). This finding suggests that this new clade of HDV-8 sequences could be classified in a new subgenotype, which we would putatively designate as 8c. Phylogenetic trees reconstructed by phylogeny software based on the maximum-likelihood principle (IQ-TREE v.1.6.12) employing two statistical support methods, aLRT and bootstrap analysis with 1,000 replicates, also supported the distinct clade with values of 87.4 and 92, respectively . Further phylogenetic and phylogeographic analysis comprising whole HDV genome and gathering previous information on sample collection date and locality would confirm or not this hypothesis.HDV-8 has been described in Gabon, Congo, Ivory Coast, and Senegal , 22, 24.l-HDAg encoding sequences , according to the manufacturer’s instructions. Partial hepatitis delta antigen (HDAg) amplification (~320 bp) was performed by RT-PCR using “Superscript III One-Step RT-PCR system”  followed by a nested PCR as previously described . DNA seqn = 65), HDV-5 (n = 16), and HDV-8 (n = 18), which were submitted to a new phylogenetic evaluation with a Bayesian Inference using the Bayesian Markov chain Monte Carlo (MCMC) statistical framework implemented in BEAST v1.10.4 software.Genotyping was performed through phylogenetic comparison employing a data set composed of 21 representative sequences of all HDV subgenotypes as suggested by Miao et al., 2019 . PhylogeON286983-ON286993. Information regarding the sequences used in the analyses are available in the supplementary table.As HDV-8 sequences available in GenBank were shorter than the sequence determined here, the multiple sequence alignment was shortened to 293 nt prior to phylogenetic analysis. Intragenotypic and intersubgenotypic diversity was evaluated by genetic distance calculation using MEGA X software . NucleotP value < 0.05 was considered statistically significant.Clinical and epidemiological data were coded and entered into a datasheet . Descriptive statistics were generated for the responses, and chi-squared test for independence or for trend and Fisher Exact Test were used to compare categorical variables according to HDV-RNA positivity in GraphPad InStat. The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Ethics Committee of Oswaldo Cruz Foundation (FIOCRUZ) under CAAE number 77364217.0.0000.5248.Informed consent was obtained from all subjects involved in the study.ON286983-ON286993. Information regarding the sequences used in the analyses are available in supplementary table.Nucleotide sequences obtained during this study were deposited in the GenBank database under accession numbers